Biological materials, such as proteins, peptides, nucleic acids, bacteria, cells, antibodies, enzymes, serums, vaccines, liposomes, and viruses, are generally unstable when stored in media or other liquid solutions. For example, enveloped viruses such as live influenza virus manufactured from egg allantoid fluid loose one log of potency, defined as Tissue Culture Infectious Dose (TCID50), in less than two to three weeks when stored under refrigerated temperature, i.e. approximately 4° C. At room temperature conditions (approximately 25° C.) and at warmer temperatures such as 37° C., the virus looses the such potency in a matter of days to hours, respectively. Bulk lyophilization processes, where aqueous formulas are frozen into solid blocks then dried by sublimation, are commonly used to stabilize these biological materials. Spray-drying is another process commonly used to remove water from biological materials to provide stability in storage. Substitution of protectant molecules, such as carbohydrates, after removal of water can increase stability by preventing chemical degradation, denaturation, and growth of microbial contaminants.
In lyophilization (freeze-drying), the biological material is commonly mixed as a solution or suspension with protective agents, frozen, and dehydrated by sublimation and secondary drying. The low temperatures of freezing and drying by sublimation can slow the kinetics of degradation reactions, but prolonged secondary drying processes carried out at elevated temperatures are often required to reduce residual moisture to an acceptable level. Moreover, freeze dried cakes must be laboriously ground and sized to a small and narrow size range if administration by inhalation is desired.
Lyophilization and secondary drying processes, as commonly practiced, can force a cell, virus, or biomolecule to undergo significant chemical and physical degradation. Degradation can be the loss of protein activity due to concentration of salts, precipitation/crystallization, shear stress, pH extremes, and residual moisture remaining through the freeze-drying. Freeze-drying can damage internal cell structures with ice crystals, fail to protect these compartments with stabilizer molecules, and destroy the bioactivity of internal molecules.
The formation of powder particles by grinding of lyophilized cakes or by spray drying is of substantial interest and importance to the biopharmaceutical industry for preservation and administration of biologically active materials. Not only can such fine particles provide a convenient storage form for biomaterials such as cells, viruses, proteins, non-protein biomolecules (including for example, DNA, RNA, lipids, and carbohydrates), but they can be substantially dehydrated for long-term storage, and rewettable for administration of the biomaterial for its intended use after the storage period. Such dried fine particles can be produced in a controlled diameter range and administered as a dried aerosol power, for example, via the pulmonary route, where the respiratory tract mucosa can rewet and dissolve the biomaterial in a patient. Numerous other uses of such fine and microfine particles containing a biomaterial are found in the art of pharmaceutics, biologics, and particularly in the field of live virus vaccines. Thus, it would be advantageous to develop methods of forming stable, specifically sized particles containing biologically active materials.
Spray drying is a well known process long used, e.g., in the food processing industry. For example, liquid products, such as milk, are sprayed through a nozzle into a stream of hot gasses to produce a powder. The increased surface area exposed in the spray mist, in combination with the high temperatures of the drying gas, can provide rapid removal of water from the liquid product. However, such process conditions are often unsuitable for sensitive biologic materials due to the shear stress, heat stress, oxidative stress, and conformational changes that can occur with loss of hydration water at high temperatures. There are several reports of spray drying therapeutic agents for pulmonary delivery, such as: Maa et al. J. Pharm. Sci. 87(2):152 (1998); Mumenthaler et al., Pharm. Res. 11(1):12 (1994); Chan et al., Pharm. Res. 14(2):431 (1997), PCT Publication No. WO 97/41833; U.S. Pat. No. 5,019,400 and WO 90/13285; Yeo et al., Biotechnology and Bioengineering 41:341 (1993) and Winters et al., J. Pharm. Sci. 85(6):586 (1996). Some of the problems encountered in spray drying pharmaceutical compositions are addressed in U.S. Pat. No. 5,902,844, Spray Drying of Pharmaceutical Formulations Containing Amino Acid-Based Materials, to Wilson. In Wilson, peptides in solution with a water soluble polymer are sprayed into a stream of drying gas to form a pharmaceutical composition. The presence of the polymer can protect the peptide from degradation by coating the peptide against chemical attacks and by substituting for water of hydration lost during drying. Certain sensitive peptides and other biological materials, such as nucleic acids, bacteria, cells, antibodies, enzymes, serums, vaccines, liposomes, and viruses can still be damaged, however, by the heat, shear stress and dehydration of the processes described by Wilson, and the like.
The heat and stress of bulk freeze drying and common spray drying can be reduced by spray freeze drying methods. For example, in U.S. Pat. No. 6,284,282, Methods for Spray Freeze Drying Proteins for Pharmaceutical Administration, to Maa et al., formulations of therapeutic proteins are atomized to into droplets that are frozen by immersion in a cold fluid before annealing and lyophilization to form particles with a physical size of from 6 um to 8 um. The particles formed by this method can be suitable for delivery of the therapeutic protein by pulmonary administration. Spray freeze drying can reduce shear stress by preparing particles with a small aerodynamic diameter from droplets with a larger physical diameter. Spray freeze drying can reduce heat stress by processing formulations in a cold environment and by providing a surface to volume ratio favorable to quick drying. However, the Maa methods are limited to protein therapeutics for pulmonary administration.
Drugs in the form of powder particles can be administered by inhalation. Inhalation therapy involves the administration of a drug in an aerosol form to the respiratory tract and includes both intranasal administration (via the upper respiratory tract including the nasal mucosa) and pulmonary administration (via the lower respiratory tract). Several means have been developed to deliver compounds directly to the passages of the lung or nose (see, pending application “Spray Freeze Dry of Compositions for Intranasal Administration”, by Vu Truong-Le, et. al., 10/412,651, filed Apr. 10, 2003, full disclosure of which is incorporated herein by reference). The most common form, especially for water-insoluble drugs, is a powder suspension that is propelled into the mouth while the patient inhales. The pulmonary deposition efficiency of powder aerosols is influenced by several factors including physical shape and size, density, porosity, and flow patterns during delivery. The particle size distribution of the aerosolized drug compositions is very important to the therapeutic efficacy of the drug when delivered by inhalation. In spray freeze drying, the size of the liquid droplet is predictive of the powder particle size such that it is often possible to control the size distribution of the powder by controlling that of the droplets. Studies of inhaled aerosols indicate that particles or droplets of greater than about 20 micrometers in mean aerodynamic diameter are effectively excluded from entry into the lungs and are captured in the nasal-pharyngeal passages. Thus, the drug compounds to be delivered to the lung are usually formulated in such a way that the median aerodynamic diameter is below about 10 micrometers. In addition, even smaller particle sizes, on the order of 0.5 to 2.5 micrometers, are needed if the drug is to reach the alveolar sacs deep in the lungs.
A need remains for methods to preserve sensitive biological materials, such as proteins and live viruses in storage, particularly at temperatures above freezing. Methods to spray freeze dry a variety of bioactive materials, including cells, viruses, bacteria and liposomes, under low shear stress conditions, for stable storage, and/or for delivery by the pulmonary route are desirable in the fields of medicine and scientific research. The present invention provides these and other features that will become apparent upon review of the following.